The present invention is directed to methods for the detection of prostate cancer in an individual by assessing prostate cells in a body fluid. The present invention is also related to commercial kits for use in the detection of prostate cancer.
In the United States prostate cancer is first in absolute incidence with an estimated 210,000 new cases in 1997, and second to lung cancer for deaths with an estimated 41,800 deaths in 1997. Parker et al., 1997, Cancer Abstracts 47(1):5-27. The incidence of prostate cancer has risen at an average annual rate of 3% per annum from 1960 to 1985. Boyle, 1997, Proceedingsxe2x80x94First International Consultation on Prostate Cancer. Prostate cancer affects older men with the rates of incidence for men in their 40""s being 1-2 per 100,000, the rates for Caucasian men in their 80""s 1,200 per 100,000 and for African American men 1,600 per 100,000. In 1900 only 25% of the United States population lived to the age of 65. Currently that number is 70%. Brody, 1985, Nature 315:463-466.
The guidelines for diagnosis of new prostate cancer adopted by the American Cancer Society in June 1997 recommends an annual prostate specific antigen (PSA) exam as well as a digital rectal exam (DRE) for men over 50 years of age and in certain instances over the age of 45. Further action should be taken only if one or both screening methodologies are abnormal. An abnormal PSA is defined as a PSA greater than 4.0 ng/ml.
Patients suspected of having prostate cancer based on a PSA test and a DRE, would then undergo additional radiographic and/or surgical tests to confirm diagnosis. These additional tests may include urology consultation, ultrasound and tissue biopsy with pathological evaluation. The cost associated with these confirmatory tests typically exceeds $5000.
The PSA test and DRE are acceptable screening methods but one of the short comings of these screening methods is the large number of falsely identified patients. A PSA European screening study biopsied 976 men and detected 190 cases of prostate cancer. 767 of the 976 men had either of both a PSA value in the range of 4 to 10 ng/ml or a suspect DRE. Of these 767 patients, only 177 had prostate cancer. 77% of the population did not have prostate cancer. Schrxc3x6der, 1997, First International Consultation on Prostate Cancer, 179-210.
Final determination of prostate cancer must be made with more expensive and invasive biopsy. However, even a single biopsy can miss prostate cancer. In one third of patients who underwent a serial biopsy study, multiple biopsies were required for the detection of prostate cancer. Keetch et al., 1994, J. Urol. 151:1571-1574.
A recent methodology attempted to further identify patients who are at risk for prostate cancer has been the determination of percent free PSA. Murphy et al., 1996, Cancer 78(4):809-818, report a difference in the ratio of free PSA to total PSA in 226 patients with benign prostatic hyperplasia and patients with no evidence of disease compared to the same ratio in patients with biopsy-proven prostate cancer. The ratio was 12.1% in patients with no evidence of prostate cancer and in the prostate cancer group the ratio was 7.1%. Despite the differences observed, the authors concluded that their data suggest that free PSA values do not provide additional diagnostic benefit compared to total PSA in screening populations in the presence of suspected cancer, post prostatectomy or in metastatic disease. Pronounced differences in the ability of available assays to detect free PSA have generated multiple cutoffs, ranging from 15-20%. Catalona et al., 1996, JAMA 274:1214-1220; Oesterling et al., 1995, J. Urol. 154:1090-1095; Toubert et al., 1996, Eur. J. Cancer 32A(12):2088-2093; Lilja, 1993, Urol. Clin. North Am. 20(4):681-686. Regardless of the potential sensitivity gained, prostate cancer will have to be confirmed with a cytological assessment. Clearly, the development of a non-invasive, cytologically based assay, able to compliment the traditional PSA and DRE, would assist the clinician in the early detection and treatment of prostate cancer without the necessity of relying on more expensive invasive means.
Citation or identification of any reference in Section 2 or any other section of this application shall not be construed as an admission that such reference is available as prior art to the present invention.
The present invention is directed to a method for the detection of prostate cancer comprising comparing the ratio of the number of prostate cells and the total number of epithelial cells in a body fluid sample, in which the ratio is elevated in individuals with prostate cancer as compared to individuals free of prostate cancer. The ratio is determined by (1) quantifying the number of prostate cells in a body fluid sample; (2) quantifying the total number of epithelial cells in the fluid sample; and (3) calculating the ratio of the number of prostate cells to the total number of epithelial cells. In an aspect of this embodiment of the present invention, the prostate cells are quantified by using an antibody which is specific for a prostate cell-specific marker, such as the prostate specific antigen (PSA) or the prostate specific membrane antigen (PSMA). In a preferred aspect, the prostate cell-specific marker is PSMA. The body fluid can be, but is not limited to, blood, urine or semen. In a preferred aspect, the body fluid is semen.
The present invention is also directed to a method for the detection of prostate cancer comprising comparing the ratio of the number of prostate cells which express a tumor associated marker and the total number of prostate cells in a body fluid sample, in which the ratio is elevated in individuals with prostate cancer as compared to individuals free of prostate cancer. The ratio is determined by (1) quantifying the total number of prostate cells in a body fluid sample; (2) quantifying the number of prostate cells in the fluid sample which express a tumor associated marker; and (3) calculating the ratio of the number of prostate cells which express the marker to the total number of prostate cells. In an aspect of this embodiment of the present invention, the prostate cells are quantified by using an antibody which is specific for a prostate cell-specific marker, such as the prostate specific antigen (PSA) or the prostate specific membrane antigen (PSMA). In a preferred aspect of this embodiment, the prostate cell-specific marker is PSMA. The body fluid can be, but is not limited to, blood, urine or semen. In a preferred aspect, the body fluid is semen.
The present invention is also directed to commercial kits for the detection of prostate cancer which comprise a prostate cell marker-specific antibody and an epithelial cell marker-specific antibody in one or more containers. Alternatively, the commercial kits comprise a prostate cell marker-specific antibody and a tumor associated marker-specific antibody in one or more containers.
As used herein, the term xe2x80x9cepithelial cell markerxe2x80x9d refers to an epithelial cell marker not present on sperm cells.
As used herein, the term xe2x80x9cprostate cell-specific antibodyxe2x80x9d refers to any polyclonal or monoclonal antibody, or portion thereof, that can immunospecifically bind to an epitope present on or in a prostate cell. For example, such an antibody immunospecifically binds to PSMA or binds to an extracellular portion or to an intracellular portion of PSMA.
As used herein, the term xe2x80x9ctumor associated marker-specific antibodyxe2x80x9d refers to any polyclonal or monoclonal antibody, or portion thereof, that can immunospecifically bind to a tumor associated marker, which when expressed in or on a cell is indicative that such cell is cancerous.
The invention may be understood more fully by reference to the following brief description of the figures, detailed description, illustrative, non-limiting examples and the appended figures.
FIGS. 1A and 1B are representative dot plots of cells binding antibody to PSMA and to cytokeratin from a prostate cancer patient (FIG. 1A) and a patient without any evidence of disease (FIG. 1B). These graphs were generated from the cycling populations as determined by DNA pulse width and area graphs. Quadrants are based on the staining of the control cells LNCAP (CAM 5.2 +, 7E11.C5 +) and PC-3 (isotype control-FITC, 7E11.C5xe2x88x92) that were run in conjunction with the samples. The prostate cancer patient sample demonstrates the 7E11.C5 positive staining of the cytokeratin population and the relative lack of staining of the cytokeratin population in the normal group.
FIGS. 2A and 2B are representative dot plots of cells from a prostate cancer patient showing the comparative PSMA and PSA staining. These graphs were generated from the cycling populations as determined by DNA pulse width and area graphs. Quadrants are based on the staining of the control cells LNCaP (CAM 5.2 7E11.C5 +, PSA +) and PNCF 007 (CAM 5.2xe2x88x92, 7E11.C5xe2x88x92, PSA xe2x88x92) that were run in conjunction with the samples. PSMA/cytokeratin staining is demonstrated in FIG. 2A (CAM 5.2 positive cells only stained positive for PSMA) and PSA/cytokeratin staining in FIG. 2B (CAM positive and CAM negative cells stained with PSA).
FIG. 3 is a graph showing the distribution of the ratio of PSMA-positive cells:cytokeratin-positive cells in subjects free of prostate cancer (NED), a subject having benign prostatic hyperplasia (BPH) and subjects having prostate cancer (CaP). Subjects who had more than one sample tested are indicated by a triangle (2 samples) or a square (5 samples). Subjects who had a single sample tested are indicated by a circle.
FIGS. 4A and 4B are graphical depictions of PSA levels (FIG. 4A) and PSMA:cytokeratin ratios (FIG. 4B) observed in a prostate cancer patient undergoing anti-androgen treatment. The time during which the patient was treated with the anti-androgen is depicted by the shaded areas labeled xe2x80x9cOn Casodexxe2x80x9d. Biopsies were taken at day 70 and 345 and the biopsy results are shown in the boxes and demonstrate the growth of the tumor over a one year period as reflected in the PSA levels and the changing PSMA:cytokeratin ratio.
FIG. 5 is a Receiver-operator characteristic plot which demonstrates that in this illustrative example the cut value above which an individual is classified as having cancer was determined to be 0.21 by maximizing the sensitivity (positive-predictive ability) and specificity (negative-predicitve ability) of the assay. The intersection of the specificity curve and the sensitivity curve is the point of maximum sensitivity and specificity. See Section 6 for details. Specificity curve, xe2x97xaf; sensitivity curve, xe2x80x94▪xe2x80x94; accuracy curve, xe2x80x94xcex94xe2x80x94.
FIG. 6A and 6B are representative dot plots of cells from a split semen sample binding antibody to PSMA and to cytokeratin with different fluorescent dyes. In FIG. 6A the PSMA antibody is coupled to the dye r-phycoerythrin (PE) via a strepavidin-biotin bridge and the DNA stain used was To-Pro 3. In FIG. 6B the PSMA antibody is linked to an alternative fluorochrome, Phycobilisome-3, and the DNA stain used was Po-Pro 3.